High pressure liquid chromatography (HPLC) is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields currently comprise only about 50% of HPLC users. However, HPLC is currently used by a variety of fields including cosmetics, energy, food, and environmental industries.
Prior to the 1970's, few reliable chromatographic methods were commercially available to laboratory scientists. During the 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flowthrough time, thus reducing purification times of compounds being isolated by column chromatography. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated.
HPLC was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In general, HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column. The analyte is forced through a column of the stationary phase by introducing a liquid at high pressure. Use of pressure prevents the components from diffusing within the column, leading to improved resolution in the resulting chromatogram. Solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as trifluoroacetic acid.
In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds, and by the 1980's HPLC was commonly used for the separation of chemical compounds. Modern HPLC has many applications including separation, identification, purification, and quantification of various compounds. Computers and automation have added to the convenience of HPLC, and the past few decades have seen a vast undertaking in the development of the micro-columns and other specialized columns which have yielded improved results.
HPLC typically comprises a pump, column, and a detector. According to Van Deep curve, for a given LC column demension and packings, there is an optimum flowrate to achieve the maximum separation efficiency or plate number.
Sensitivity and resolution are the most important indicators in chromatography. Sensitivity depends on several factors. One of the most important factors is the residence time (or Time of Flight. TOF) of a peak passing through the detector. Resolution also depends on the duration the peak passing through the detector. Therefore, the detector's flow cell volume is selected to balance between resolution and sensitivity requirements. If the flow cell is too large, the sensitivity increases, however, the resolution will be sacrificed. If the flow cell is too small, then the resolution increases, but the sensitivity decreases.
Simply slowing the LC flowrate will deviate from optimum separation efficiency. Furthermore, under normal settings, slowing down HPLC flowrate would result in lower pressure in column.
Pressure is one of the most important factors in HPLC separation. Interruption of pressure during the separation would result in poor separation performance and an unusual detector response (Macko et al., J. Liq. Chromtography Rel. Tech., 2001, 24, 1275-1293).
However, the traditional methods do not have a mechanism to prevent the column pressure drop when LC flowrate change occurs.
The conflict between sensitivity and resolution is apparent for radioactivity detection in HPLC. In order to detect radioactivity from HPLC, a flow cell with either a solid or liquid scintillator is used. In case of a liquid cell, a separate pump is used to deliver liquid scintillator to be used with eluate from HPLC and the mixture flows through the flow cell, normally made of a coiled transparent tubing. When using a solid cell, the eluate pass through the flow cell packed with solid scintillator particles.
To detect radioactivity from HPLC, a flow cell with either a solid or liquid scintillator is used. In case of a liquid cell, a separate pump is used to deliver liquid scintillator to be used with eluate from HPLC and the mixture flows through the flow cell, normally made of a coiled transparent tubing. When using a solid cell, the eluate pass through the flow cell packed with solid scintillator particles. The conflict between detection sensitivity and signal resolution is also fundamental to radioactivity detection in HPLC.
Many advances in HPLC technology have been made in the past few decades, however, currently available detectors are unable to effectively detect/process the eluting peaks, especially when the peak width is getting smaller with faster separation in HPLC columns. The time a peak flows through a flow cell is called TOF (Time-of-Flight). TOF depends on several parameters: LC flowrate, agent flowrate, and cell volume. For example, under normal circumstance when LC flowrate is 1 ml/min, volume-adding component flows at 3:1 ratio, a flow cell of 500 μl volume is used to obtain optimum sensitivity with good resolution. This setting will result in a TOF of (500/(1000/(1+3))×60)=7.5 seconds. The TOF is very important because TOF affects the peak resolution and detection sensitivity. If TOF is too long, then the peaks can not be resolved well and the separation is compromised. In the other words, if TOF is too long, two narrowly resolved peaks might end up residing in one cell volume so that they are detected as one merged peak. On the other hand, when TOF is too small, the radioactivity detection sensitivity degrades since the quantity of radioactivity detection depends on the counting time or TOF. For radioactivity detection, the longer the counting time (or TOF), the better accuracy the detection is. Under these conditions, all the peaks will be counted for the same or similar TOF and the sensitivity for smaller peaks will be low. Smaller peaks can not be detected and missed due to the poor sensitivity. In order to achieve high sensitivity of radioactivity detection, traditionally LC fractions are collected for off-line counting for longer period of time. Recently, a on-line stop-flow technique is being used to increase the radioactivity detection sensitivity (U.S. Pat. No. 6,546,786; and Nassar et al., Anal. Chem., 2003, 75, 785-790). Those methods result in longer analytical time. Thus, there is a need for a better system that can preserve optimum separation of analyte components and provide a longer time for detector analysis to simultaneously increase detection sensitivity and signal resolution.